Serum chemokines in patients with rheumatoid arthritis treated with etanercept

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1 Serum chemokines in patients with rheumatoid arthritis treated with etanercept Piotr Adrian Klimiuk, Stanislaw Sierakowski, Izabela Domyslawska, Justyna Chwiecko To cite this version: Piotr Adrian Klimiuk, Stanislaw Sierakowski, Izabela Domyslawska, Justyna Chwiecko. Serum chemokines in patients with rheumatoid arthritis treated with etanercept. Rheumatology International, Springer Verlag, 2009, 31 (4), pp < /s >. <hal > HAL Id: hal Submitted on 6 Apr 2011 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2 Serum chemokines in patients with rheumatoid arthritis treated with etanercept Piotr Adrian Klimiuk, Stanislaw Sierakowski, Izabela Domyslawska, Justyna Chwiecko Department of Rheumatology and Internal Diseases, Medical University of Bialystok, Bialystok, Poland Piotr Adrian Klimiuk, MD, PhD, Assistant; Stanislaw Sierakowski, MD, PhD, DSc (Med.), Professor and Chairman; Izabela Domyslawska, MD, PhD, Assistant; Justyna Chwiecko, MA, PhD, Principal Technician. Please address correspondence and reprint requests to: Dr. Piotr A. Klimiuk, Department of Rheumatology and Internal Diseases, Medical University of Bialystok, M.C. Sklodowskiej 24a, Bialystok, Poland. Telephone: , Fax: , Running title: Etanercept and chemokines in RA

3 2 Abstract Objective. Chemokines promote leukocyte traffic into the synovium, leading to the initiation and progression of the rheumatoid arthritis (RA). The aim of the study was to determine the effects of etanercept, a soluble tumour necrosis factor receptor (stnfr), on the serum chemokines levels in patients with active RA. Methods. Patients were treated with 50mg of subcutaneous injection of etanercept per week and methotrexate (10-25mg/week). Serum levels of interleukin-8 (IL-8), RANTES (regulated upon activation, normal T cell expressed and secreted) and monocyte chemoattractant protein- 1 (MCP-1) were assessed by ELISA at months 0, 3, 6, 9 and at month 12, prior to injection. Results. Three months treatment with etanercept diminished serum concentrations of IL-8, RANTES and MCP-1 (p<0.05, p<0.01 and p < 0.001, respectively). Subsequent etanercept administrations prolonged decrease in serum chemokines levels and in the case of IL-8 even intensified the reduction of its concentration in serum. These changes were accompanied by significant decrease of disease activity score (DAS28) (in all cases p<0.001). Prior to the first etanercept administration serum concentrations of studied chemokines correlated with markers of RA activity such as the erythrocyte sedimentation rate (ESR) and DAS28. Following next drug injection such associations were less or not significant. Conclusion. Therapy with etanercept and MTX, not only caused a clinical improvement, but also diminished serum chemokines levels in RA patients. Further treatment with etanercept sustained chemokines suppression. Key words: chemokines, IL-8, RANTES, MCP-1, rheumatoid arthritis, etanercept.

4 3 Introduction Rheumatoid arthritis (RA) is characterized by infiltration of mononuclear cells into synovium. Enhanced angiogenesis and the proliferation of the synovium-lining layer may also be noticed. Macrophages, fibroblasts, lymphocytes and endothelial cells in rheumatoid synovium are supposed to contribute to the pathogenesis of the disease by several mechanisms including chemokines production [1, 2]. Chemokines have an important role in the migration of mononuclear cells into synovium, leading to the initiation and progression of the disease. These small proteins enhance the inflammation by recruiting and activating leukocyte subpopulations. Furthermore, chemokines like interleukin-8 (IL-8) or monocyte chemoattractant protein-1 (MCP-1) intensify infiltration of synovial tissue by inflammatory cells through the stimulation of neovascularisation of rheumatoid synovium [3, 4]. Chemokine production is stimulated by inflammatory cytokines like interleukin-1 (IL-1) and tumour necrosis factor α (TNF-α) [5, 6]. In addition, chemokines promote synthesis of proinflammatory cytokines like IL-1, IL-6, TNF-α or matrix metalloproteinases engaged in the joint destruction in RA [3, 7]. Abundant presence of such chemokines like IL-8, RANTES (regulated upon activation, normal T cell expressed and secreted) or MCP-1 in rheumatoid synovium even in early stage of the disease, support the role of chemokines in RA [8, 9, 10]. Several studies have shown that anti-tnf-α treatment with infliximab, a chimeric antitumour necrosis factor α antibody [11, 12] and etanercept, a soluble tumour necrosis factor receptor (stnfr) [13, 14, 15] diminish clinical activity of RA. In our previous report, we demonstrated that infusions of infliximab reduce serum chemokines concentrations in RA patients [16]. In the present study, we investigated the effect of the one-year therapy with etanercept on serum IL-8, RANTES and MCP-1 serum levels in patients with active rheumatoid arthritis.

5 4 Materials and methods Patients and samples Eligible patients (3 men and 14 women) met the American College of Rheumatology 1987 revised diagnostic criteria for RA [17]. At the time of enrolment, patients had active RA, as manifested by at least 6 tender and 6 swollen joints, and two of the following: morning stiffness for more than 45 minutes, C-reactive protein (CRP) level of more than 20 mg/l, erythrocyte sedimentation rate (ESR) of more than 28 mm/h. None of the patients had previous history of tuberculosis or symptoms of infectious diseases in the previous 3 months. Chest x-rays performed prior to the first etanercept administration were normal in all patients. Mean patients age was 41.6 years (range years), and mean disease duration was 5.1±4.4 years. All subjects were receiving methotrexate (MTX) (median 17.5 mg/week, range mg/week) in a stable dose for at least 2 months and nonsteroidal anti-inflammatory drugs (NSAIDs) in a stable dose for at least 4 weeks before enrolment into the study. Fourteen patients were receiving corticosteroids (median 7.5 mg/day of prednisone or equivalent, range 5-10mg/day) in a stable dose for at least 4 weeks before beginning of the study. Such treatment regimen with MTX, NSAIDs and corticosteroids was maintained throughout the study. Patients were receiving etanercept, a soluble tumour necrosis factor receptor (stnfr), 50mg subcutaneously weekly during 12 months. Clinical and laboratory assessment were conducted prior to injection at baseline and at months 3, 6, 9, and 12. Blood samples were clotted for 30 minutes and next centrifuged for 15 minutes at 1000 x g. Serum aliquots were stored at -80 C until assayed. The local ethical committee approved the study protocol and all patients provided written informed consent.

6 5 Clinical and laboratory measures The evaluation included the number of tender joints (of 28 joints assessed), the number of swollen joints (of 28 assessed), three variables disease activity score that include 28-joint counts (DAS28) [18], the duration of morning stiffness, blood cell counts, including differential white blood counts, erythrocyte sedimentation rate (ESR), rheumatoid factor level, transaminases activity and creatinine concentration in serum. Measurements were performed at baseline and at months 3, 6, 9 and 12 (prior to etanercept injection). Enzyme-linked immunosorbent assays (ELISA) The assessments of serum levels of studied chemokines (IL-8, RANTES, MCP-1) were determined by ELISA kits from R&D Systems, Wiesbaden-Nordenstadt, Germany, strictly according to the manufacturer s instructions. The sensitivity of the assays was 3.5 pg/ml (IL- 8), 2 pg/ml (RANTES) and 5 pg/ml (MCP-1). Statistical analysis The normally distributed data were analyzed by paired Student t-test. Wilcoxon signed rank test was used to compare the differences between non-normally distributed data. Correlations between variables were assessed by Spearman rank order test. P values lower than 0.05 were considered statistically significant. Results Serum concentrations of chemokines Etanercept decreased serum concentrations of IL-8 after three months of treatment as compared to baseline values (p<0.05) (Figure 1). Continuation of therapy even more significantly diminished serum IL-8 levels as assessed at months 6, 9 and 12 (in all cases p<0.001). Down regulation of IL-8 was accompanied by the fall of serum concentrations of

7 6 RANTES as evaluated at months 3, 6, 9 and 12 (p<0.01, p<0.001, p<0.001 and p<0.01 respectively) (Figure 2). Three months treatment with etanercept suppressed also MCP-1 serum level (p<0.001) (Figure 3). Subsequent drug administrations prolonged MCP-1 reduction, although less significantly (p<0.01). Correlations between serum levels of chemokines and clinical data Down regulation of circulating chemokines was accompanied by significant decrease of three variables disease activity score that include 28-joint counts (DAS28) as assessed at months 3, 6, 9 and 12 (in all cases p<0.001) (data not shown). At baseline we demonstrated significant correlations between IL-8 and RANTES serum concentrations, and such markers of RA activity as the erythrocyte sedimentation rate (ESR) or DAS28 (in all cases p<0.01) (Table 1). Furthermore we observed significant correlations between MCP-1 serum levels and ESR or DAS28 (p<0.01 and p<0.05 respectively). During therapy with etanercept such associations were less or not significant. No associations between patient sex, age or disease duration with studied circulating chemokines were observed. Discussion Chemokines are proteins, which work as mediators of inflammation process by recruiting and activating particular leukocyte populations. Along with adhesion molecules, they control the migration of mononuclear cells into synovium, leading to the initiation and progression of the rheumatoid arthritis. Chemokine synthesis is regulated by inflammatory cytokines like interleukin-1 (IL-1) and tumour necrosis factor α (TNF-α) [5, 6]. Previously we revealed that another blocker of TNF-α, a chimeric anti-tnf-α monoclonal antibody (infliximab), not only suppress clinical activity of RA, but also down regulate serum levels of IL-8, RANTES and MCP-1 [16]. In current study, we analysed serum concentrations of IL-8, RANTES and

8 7 MCP-1 in patients with active RA treated with etanercept, a soluble tumour necrosis factor receptor (stnfr). IL-8 is a member of CXC chemokine family and is produced by different cells like macrophages, fibroblasts, T cells, neutrophils, endothelial cells and chondrocytes [3, 6]. Elevated IL-8 levels were found not only in serum [19, 20] but also in synovial fluid [20] of RA patients compared to patients with OA or in healthy controls. In our present study, we demonstrated that treatment with etanercept decreased serum levels of IL-8 in RA patients. Especially serum concentrations of that chemokine were reduced after 6 months of therapy and afterwards. Prior to etanercept administration serum IL-8 was correlated with clinical markers of disease activity like erythrocyte sedimentation rate (ESR) and disease activity score (DAS28). Circulating IL-8 was demonstrated by other investigators to correlate with serum MCP-1 [5]. We also observed the association between serum levels of IL-8 and MCP- 1. Moreover, we noted the correlation between serum IL-8 and RANTES concentrations prior to the beginning of etanercept treatment. However, after subsequent drug injections such associations were less or not significant. RANTES (regulated upon activation, normal T cell expressed and secreted) belongs to CC chemokine family, produced by T cells and synovial fibroblasts [21, 22], and involved in monocyte and T lymphocyte migration [3, 23]. Enhanced concentrations of RANTES were found in RA serum [19, 24] and synovial fluid [25] compared to OA patients and healthy individuals. In rheumatoid arthritis this chemokine was demonstrated to be predictive of radiological erosions [26]. In our study we showed that therapy with etanercept reduce serum levels of RANTES in RA patients. Further drug administrations prolonged circulating RANTES reduction in studied patients. Prior to the etanercept therapy serum RANTES concentrations were demonstrated by us to correlate with ESR and DAS28. Furthermore, serum RANTES was associated also with IL-8 and MCP-1 levels. As in the case of IL-8, after further etanercept administrations such correlations were less or not significant.

9 8 MCP-1 (monocyte chemoattractant protein-1) is produced by leucocytes, fibroblasts, endothelial cells and chondrocytes and is member of CC chemokine family [5, 6]. It attracts not only monocytes, but also T cells, natural killer cells and basophils, and plays a role in T cell differentiation and angiogenesis [3]. Elevated MCP-1 levels were revealed in serum and synovial fluid of RA patients [19, 27, 28]. In the current study, serum MCP-1 levels were down regulated following 3 months therapy with etanercept. Further drug injections maintained MCP-1 suppression, however to a lesser extent. Also others demonstrated serum MCP-1 levels reduction in RA patients during 6 months treatment with etanercept [29]. Other investigators correlated serum levels of MCP-1 with IL-8 concentrations [5], swollen joint count and Ritchie articular index [30] in RA. In our analysis prior to etanercept administrations circulating MCP-1 was correlated with clinical markers of disease activity like ESR and disease activity score (DAS28). Furthermore, we also noted the associations between serum concentrations of MCP-1 and IL-8 or RANTES concentrations. Following later on etanercept administrations these associations were less or not significant. We found no correlations between patient sex, age or disease duration with serum concentrations of studied chemokines. As in other studies [13, 14, 15] parameters of disease activity such as ESR and disease activity score (DAS28) decreased after three months therapy with etanercept. Subsequent drug administrations caused further decrease of these parameters of disease activity (data not shown). In conclusion, three months etanercept treatment combined with MTX, beside a rapid clinical improvement, down regulated serum IL-8, RANTES and MCP-1 levels in RA patients. Continued administrations of etanercept prolonged reduction of studied chemokines during one-year study period. Moreover, we revealed that serum concentrations of IL-8, RANTES and MCP-1 correlated with markers of disease activity such as ESR and the disease activity score (DAS28) prior to the first etenarcept administration and to a lesser extent

10 9 following further drug injections. Therefore, these chemokines might be useful markers of the RA activity in patients with the anti-tnf-α therapy. Key messages Therapy with etanercept and MTX cause a clinical improvement and diminish serum chemokines levels in rheumatoid arthritis (RA) patients during one-year observation. Serum concentrations of IL-8, RANTES and MCP-1 might be useful markers of the RA activity in patients with the anti-tnf-α therapy. Conflict of interest statement: None Acknowledgements Poland. Study was support by the grant ( L) from Medical University of Bialystok,

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15 14 Legends for illustrations Figure 1. Serum concentrations of interleukin-8 (IL-8) in rheumatoid arthritis (RA) patients, assessed by ELISA technique. Patients were treated with 50mg of subcutaneous injection of etanercept per week. Blood samples were obtained at months 0, 3, 6, 9 and 12 prior to injection. Box plots represent median (line), 25th and 75th percentiles (box), and 10th and 90th percentiles (whiskers). Significance of differences between pre-injection IL-8 values at month 0 and following months were expressed as: *p<0.05, **p<0.01, ***p< Significance of differences between pre-injection values at month 3 and following months were expressed as: # p<0.05. Figure 2. Serum concentrations of RANTES (regulated upon activation normal T cell expressed and secreted) in RA patients, determined and presented as described in the legend to Figure 1. Figure 3. Serum concentrations of monocyte chemoattractant protein-1 (MCP-1) in RA patients, determined and presented as described in the legend to Figure 1. Table 1. Correlations between serum concentrations of chemokines and DAS28 in RA patients studied. RANTES MCP-1 ESR DAS28 IL ** 0.564* 0.703** 0.681** RANTES 0.551* 0.647** 0.691** MCP ** 0.559*

16 15 RA: rheumatoid arthritis, IL-8, interleukin-8; RANTES, regulated upon activation normal T cell expressed and secreted; MCP-1, monocyte chemoattractant protein-1; ESR: erythrocyte sedimentation rate; DAS28: three variables disease activity score that include 28-joint count. Data expressed as r values (correlation coefficient) according to Spearman rank correlation: * p<0.05, ** p<0.01 DAS28 evaluations were performed, and blood samples were obtained on month 0, prior to first etanercept injection. 60 * *** # *** # *** IL-8 (pg/ml) Months

17 16 RANTES (ng/ml) ** *** *** ** Months

18 *** ** ** ** MCP-1 (pg/ml) Months